Leptospirosis is a global, zoonotic disease caused by members of the genus Leptospira. Although widespread and sometimes fatal, leptospirosis is considered a neglected and understudied disease. The causative agent of Leptospirosis was first identified 100 years ago in 1916, but the slow in vitro growth rate and limited genetic tools available to manipulate the genome of this spirochete have hampered the identification of virulence factors and development of a vaccine. Leptospires can be broadly divided into two groups: free-living saprophytes and infectious pathogens. The most widely used and studied species are L. biflexa (a non-pathogenic saprophyte) and L. interrogans (a pathogen). However, the non-pathogenic L. biflexa is more easily cultivated and more amenable to genetic manipulation than the pathogenic L. interrogans. Therefore, we have focused on L. biflexa as a model to understand the genus as a whole, develop new techniques, and as a heterologous host to express pathogen-specific genes in order to characterize their function. Targeted gene inactivation, shuttle vector transformation, and transposon mutagenesis have all been successfully used in L. biflexa. To date, there are few published reports of targeted gene inactivations in L. interrogans. Transposon mutagenesis can be applied to L. interrogans but it functions at such a low efficiency that it cannot be utilized for any broad applications, such as auxotrophic screens or signature tagged mutagenesis. Since L. biflexa has a better transformation frequency than other species, we plan to optimize new techniques in this organism. In FY2016, we completed a proteomic map of in vitro cultivated L. biflexa (Stewart et al. Appl. Environ. Microbial. 82:1183-95). This project identified highly abundant proteins from membrane- and soluble-fractions that can be used as cellular markers, as controls for gene expression studies, and also quantified the transcript data from a subset of these genes. Further, we demonstrated that a significant number of L. biflexa proteins are subject to post-translational modifications including phosphorylation, methylation, and acetylation. Highly expressed proteins allow us to identify targets that may play important physiological roles and also use as tagged proteins for various expression studies. This work was completed with internal collaborations involving Dr. James Carroll in the Laboratory of Persistent Viral Diseases, NIAID, and Daniel Sturdevant and Dr. Lisa Olano of the Research Technologies Branch, NIAID. The data generated from the proteomic study identified a bactofilin homolog in L. biflex; members of this protein family have been shown in other bacteria to contribute to morphology. In FY2016, post-bacculaureate Katrina Jackson generated a bactofilin deletion mutant and an overexpression mutant and demonstrated that this bactofilin homolog contributes to the helical structure of L. biflexa cells. We are currently characterizing the bactofilin's contribution to cellular physiology and its physical location in the cell. As this protein has a homolog in the pathogenic L. interrogans, we believe that identifying and characterizing the factors that contribute to morphology in Leptospira spp. should aid us in understanding the basic cellular physiology of these organisms and identify factors that may limit their ability to infect and disseminate in mammalian hosts. In FY2016 we have continued to evaluate different systems that may improve the transformation effiencies of leptospires. The CRISPR/Cas system, present in L. interrogans but absent in L. biflexa, targets and degrades foreign DNA and we hypothesize that it may contribute to the lower transformation frequency observed in the pathogen relative to the saprophyte. Specifically, we have analyzed the 21 complete Leptospira genomes available in the NCBI database and shown that only members of the pathogenic spp. possess CRISPR/Cas systems and these systems are absent from the saprophytic spp. Currently, we are attempting to inactivate specific cas genes in L. interrogans and move the entire operon into L. biflexa. Finally, conjugation between Escherichia coli and Leptospira spp. was recently demonstrated to be an effective method for DNA transfer between these species. However, the existing vector for conjugative transfer was capable of replicating in limited strains of E. coli. Therefore, in FY2016 we engineered a new conjugative vector capable of replicating to high copy number in any E. coli strain. We are currently using this vector for the goals described above. The long-term objective of this project is to use the improved tools and techniques to understand the basic physiology of leptospires and the mechanisms of infection and pathogenecity of L. interrogans. Together this knowledge should help accelerate the development of preventative measures against Leptospirosis.